Essay --

DNA isolation and elaborateness of ansA1 and ansA3 genesGood reference genomic DNA was isolated (Sambrook, et al 2002) and bothansA1 and ansA3 genes were amplified by PCR. Clear pecks of both the genes covering a size of 1kb were observed nether UV transilluminator after agarose mousse electrophoresis (Fig 1).Figure 1 PCR amplification of both ansA1 and ansA3 genes passageway (A) shade up 100bp marker, Lane (B) amplification of ansA1 gene, Lane (C) amplification of ansA3 geneOverexpression and cultureThe overexpressed recombinant proteins (rBliAI and rBliAIII) of B. licheniformis MTCC 429 were purified to homogeneity by affinity chromatography apply Ni-NTA column. Overexpression of recombinant His-tagged asparaginase into E.coli leads to improvement in yield and affinity purification turn of enzyme (Enriquez et al, 2012). The fractions showing presence of protein with the athletic supporter of Bradfords reagent were pooled together. The pooled protein solution was dialyze d against the same caramel brown and was checked for the L-asparaginase activity.The purified protein after SDS page or Native paginate showed a single band (Fig 2) illustrating its homogeneous nature. The molecular weight of the subunits of ansA1 and ansA3was found to be approximately 37kD after SDS PAGE analysis. SDS PAGE analysis of recombinant L-asparaginase from Pyrococcus furiosus displayed single 37kDa band (Bansal et al, 2010). In an other turn over, recombinant ansA from genus Rhizobium etliin SDS-PAGE showed the presence of a single polypeptide twine of 47kDa (Enriquez et al, 2012).Native PAGE analysis showed the molecular weight of the purified protein as 74 kDa. This study confirmed that the ansA1 and ansA3 enzymes from Bacillus licheniformis MTCC 429 atomic number 18 a homodimer in... ...81.50x106Roth et al, 2013Table Comparative energizing parameters for hydrolysis of L-asparagineThe kinetic properties of recombinant BliA were investigated and the parameters l ike kcat and kcat/Km were also studied with L-asparagine as a substrate. Comparative studies of the rBliA with asparaginases from various microbial sources showed that rBliA has depress Km value for L-asparagine (Table 2) electropositive thatrBliA hasbetter affinity towards its substrate than other reported (Table) microbial sources. The catalytic unalterable (kcat) of rBliA was only 1.5 times high than the L-asparaginase from E.coli and catalytic constant of rErAII was 1.35 times higher(prenominal) than rBliA. The absolute value of kcat/Km suggests the catalytic efficiency of the enzyme (Price and Nairn, 2009). kcat/Km of the rBliAIII was 5.4x 104 which is lower than the asparaginases from E.coli and Erwinia. Essay -- DNA isolation and amplification of ansA1 and ansA3 genesGood quality genomic DNA was isolated (Sambrook, et al 2002) and bothansA1 and ansA3 genes were amplified by PCR. Clear bands of both the genes showing a size of 1kb were observed under UV tr ansilluminator after agarose gel electrophoresis (Fig 1).Figure 1 PCR amplification of both ansA1 and ansA3 genes Lane (A) Step up 100bp marker, Lane (B) amplification of ansA1 gene, Lane (C) amplification of ansA3 geneOverexpression and purificationThe overexpressed recombinant proteins (rBliAI and rBliAIII) of B. licheniformis MTCC 429 were purified to homogeneity by affinity chromatography using Ni-NTA column. Overexpression of recombinant His-tagged asparaginase into E.coli leads to improvement in yield and affinity purification process of enzyme (Enriquez et al, 2012). The fractions showing presence of protein with the help of Bradfords reagent were pooled together. The pooled protein solution was dialyzed against the same buffer and was checked for the L-asparaginase activity.The purified protein after SDS PAGE or Native PAGE showed a single band (Fig 2) illustrating its homogeneous nature. The molecular weight of the subunits of ansA1 and ansA3was found to be approximately 37 kD after SDS PAGE analysis. SDS PAGE analysis of recombinant L-asparaginase from Pyrococcus furiosus displayed single 37kDa band (Bansal et al, 2010). In another study, recombinant ansA from Rhizobium etliin SDS-PAGE showed the presence of a single polypeptide chain of 47kDa (Enriquez et al, 2012).Native PAGE analysis showed the molecular weight of the purified protein as 74 kDa. This study confirmed that the ansA1 and ansA3 enzymes from Bacillus licheniformis MTCC 429 are a homodimer in... ...81.50x106Roth et al, 2013Table Comparative kinetic parameters for hydrolysis of L-asparagineThe kinetic properties of recombinant BliA were investigated and the parameters like kcat and kcat/Km were also studied with L-asparagine as a substrate. Comparative studies of the rBliA with asparaginases from various microbial sources showed that rBliA has lower Km value for L-asparagine (Table 2) confirming thatrBliA hasbetter affinity towards its substrate than other reported (Table) microbial sou rces. The catalytic constant (kcat) of rBliA was only 1.5 times higher than the L-asparaginase from E.coli and catalytic constant of rErAII was 1.35 times higher than rBliA. The absolute value of kcat/Km suggests the catalytic efficiency of the enzyme (Price and Nairn, 2009). kcat/Km of the rBliAIII was 5.4x 104 which is lower than the asparaginases from E.coli and Erwinia.